The retinoblastoma family of pocket proteins (pRB) is a central node impossible to bypass during the cell cycle. Dynamic variation in the ratios of all three hyper-phosphorylated paralogs is directly correlated with cell proliferation, cellular quiescence, and apoptosis, in nearly all cells in the body. Compared to other tissues, the biochemical and molecular pathways of the pRBs in the inner ear are relatively unexplored. Likewise, the effects of quantitative manipulation of this central node in the inner ear sensory epithelia cells and its potential to promote hair cell (HC) and supporting cell (SC) proliferation is yet to be determined. Establishing therapeutic measures that slow the progression of hearing loss or restoration of lost hearing are major clinical challenges. Differentiated HCs are especially susceptible to inherited and environmental pathogenic insults. Mammalian HCs proliferate during embryogenesis, then exit the cell cycle, differentiate and become functionally mature. Adult mammalian HCs do not regenerate and HC death leads to irreversible neurosensory hearing loss and balance impairment. Recent advances have provided proof of principle for two sets of therapies: the use of the cyclin system or pocket protein gene (Rb1) to promote proliferation, and the effectiveness of Atoh1 to induce transdifferentiation (TD) SC into HC. Combined, these two approaches can mimic the ability of lower vertebrates to regenerate HC. However, beyond the proof of principle, current attempts to regulate cell cycle through genetic ablation of Rb1 are not likely to safely repopulate lost HC and SC. Preliminary assessment of the three pRB members, Rb1, Rbl1 (p107) and Rbl2 (p130) revealed that all of them are expressed in the inner ear and exhibit extensive differences and similarities with one another along and across the organ of Corti. Deletion of any pRB leads to additional rows of HC and SC. Using various conditional and complete null mutants, we will investigate the role of all three pRBs in the ear and determine the ability of SCs to proliferate as a result of quantitative manipulation of the inherent ratio of the pRBs in the inner ear sensory epithelia (aim 1). Additionally, we will analyze pRBs-mediated proliferation in congenitally deaf (dreidel) mice (aim 2). Results obtained from our proposed study will provide insights on the therapeutic applicability of pRB manipulation in SC proliferation and HC regeneration. Relevance: Adult mammalian inner ear sensory hair cells (HC) do not regenerate and their death leads to irreversible neurosensory hearing loss and balance impairment. Our research project aims to apply current approaches to quantitatively manipulate the dosage of genes involved on cell proliferation and stimulate HC regeneration.